trichomes of tobacco excrete zinc as zinc-substituted...

Trichomes of Tobacco Excrete Zinc as Zinc-SubstitutedCalcium Carbonate and OtherZinc-Containing Compounds1[W]

Geraldine Sarret, Emiko Harada, Yong-Eui Choi*, Marie-Pierre Isaure, Nicolas Geoffroy, Sirine Fakra,Matthew A. Marcus, Mandy Birschwilks, Stephan Clemens, and Alain Manceau

Environmental Geochemistry Group, Laboratoire de Geophysique Interne et Tectonophysique, University ofGrenoble and Centre National de la Recherche Scientifique, 38041 Grenoble cedex 9, France (G.S., E.H., M.-P.I.,N.G., A.M.); Division of Forest Resources, College of Forest Sciences, Kangwon National University, Chunchon200–701, Kangwon-do, Korea (E.H., Y.-E.C.); Leibniz-Institut fur Pflanzenbiochemie, 06120 Halle (Saale), Germany(E.H., M.B., S.C.); and Advanced Light Source, Berkeley Lab, MS 6–2100, Berkeley, California 94720 (S.F., M.A.M.)

Tobacco (Nicotiana tabacum L. cv Xanthi) plants were exposed to toxic levels of zinc (Zn). Zn exposure resulted in toxicity signsin plants, and these damages were partly reduced by a calcium (Ca) supplement. Confocal imaging of intracellular Zn usingZinquin showed that Zn was preferentially accumulated in trichomes. Exposure to Zn and Zn 1 Ca increased the trichomedensity and induced the production of Ca/Zn mineral grains on the head cells of trichomes. These grains were aggregates ofsubmicrometer-sized crystals and poorly crystalline material and contained Ca as major element, along with subordinateamounts of Zn, manganese, potassium, chlorine, phosphorus, silicon, and magnesium. Micro x-ray diffraction revealed that thelarge majority of the grains were composed essentially of metal-substituted calcite (CaCO3). CaCO3 polymorphs (aragonite andvaterite) and CaC2O4 (Ca oxalate) mono- and dihydrate also were identified, either as an admixture to calcite or in separategrains. Some grains did not diffract, although they contained Ca, suggesting the presence of amorphous form of Ca. Thepresence of Zn-substituted calcite was confirmed by Zn K-edge micro-extended x-ray absorption fine structure spectroscopy.Zn bound to organic compounds and Zn-containing silica and phosphate were also identified by this technique. Theproportion of Zn-substituted calcite relative to the other species increased with Ca exposure. The production of Zn-containingbiogenic calcite and other Zn compounds through the trichomes is a novel mechanism involved in Zn detoxification. Thisstudy illustrates the potential of laterally resolved x-ray synchrotron radiation techniques to study biomineralization and metalhomeostasis processes in plants.

Smoking of tobacco (Nicotiana tabacum) leaves is oneof the principal routes of exposure to heavy metals.Metals contained in tobacco leaves originate from rootuptake and transfer to the shoots and also fromdeposition of aerosol particles on the leaves (Fleisherand Parungo, 1974). Efforts have been made to min-imize toxic metal contents in the leaves (Lugon-

Moulin et al., 2004). However, this is not the case forcounterfeit tobacco products, which contain higherconcentrations of metals than their genuine equiva-lents (Stephens et al., 2005). Besides its primary appli-cation for leaf production, tobacco is also a candidatefor phytoextraction. Although this plant species is nota hyperaccumulator, it has several advantages, includ-ing a high biomass, moderate soil requirements, fastgrowth rate, and ease of harvesting. For this particularapplication, research is conducted with the aim ofenhancing metal accumulation in the shoots (Songet al., 2003).

Little is known about the mechanisms of metalsaccumulation and detoxification in tobacco. The expo-sure of plants to high concentrations of metals gener-ally induces a stress, whose visible symptoms includean inhibition of root elongation, a decrease in shootgrowth, leaf chlorosis, and necrosis of the tissues. Thisstress is generated by the perturbation of cell metab-olism by metals, mostly due to the inactivation ofproteins by adventitious binding. Plants have devel-oped various mechanisms to tolerate heavy metals intheir tissues (Kupper and Kroneck, 2005). One of themost important mechanisms is the sequestration ofheavy metals in specific subcellular compartments ofparticular tissues, whose nature depends on the plant

1 This work was supported by the BioGreen 21 Program, RuralDevelopment Administration, in Korea. The operations of the Ad-vanced Light Source at Lawrence Berkeley National Laboratory aresupported by the Director, Office of Science, Office of Basic EnergySciences, Materials Sciences Division, of the U.S. Department ofEnergy (contract no. DEAC03–76SF00098). E.H. is the recipient offellowships from the BioGreen 21 program, the International HumanFrontier Science Program Organization, and the Alexander vonHumboldt Foundation.

* Corresponding author; e-mail [email protected]; fax 82–33–252–8310.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Yong-Eui Choi ([email protected]).

[W] The online version of this article contains Web-only data.Article, publication date, and citation information can be found at

www.plantphysiol.org/cgi/doi/10.1104/pp.106.082743.

Plant Physiology, July 2006, Vol. 141, pp. 1021–1034, www.plantphysiol.org � 2006 American Society of Plant Biologists 1021

species. Recently, we showed that the trichomes oftobacco exposed to Cd21 and Ca21 produced calcium(Ca)/cadmium (Cd)-containing grains (Choi et al.,2001, 2004; Choi and Harada, 2005). Other effects ofCd exposure were a retardation of plant growth and a2-fold increase of the number of trichomes in compar-ison with untreated plants. Also, an increased concen-tration of Ca in the nutrient medium had a protectiveeffect toward Cd toxicity and enhanced the productionof the grains.

Trichomes are specialized unicellular or multicellu-lar structures derived from the epidermal cell layer,which may have various functions depending on theplant species and organ (Rodriguez et al., 1983; Wagneret al., 2004). Tobacco has multicellular glandular tri-chomes of two types, the so-called long and shorttrichomes. The long trichomes are known to excretevarious alkaloids, including nicotine, whereas the shortones excrete terpenoids (resins; Hallahan et al., 2000)and defensive proteins (Shepherd et al., 2005). The Ca/Cd-containing grains were 20 to 150 mm in diameterand formed on head cells of both the short and longtrichomes. They consisted in aggregates of micrometer-sized particles. Scanning electron microscopy coupledwith energy dispersive x-ray fluorescence (SEM-EDX)analysis indicated the presence of minor amounts ofmagnesium (Mg), phosphorus (P), sulfur (S), andmanganese (Mn), in addition to major amounts of Caand Cd. Thus, these studies revealed a new function oftobacco trichomes: the excretion of Cd in the form ofparticles.

Other epidermal structures, the salt glands of Armeriamaritima (Neumann et al., 1995), Avicennia marina(MacFarlane and Burchett, 1999), and Silene vulgaris(Bringezu et al., 1999), have been shown to excrete Ca/metal-containing grains. In contrast, nonglandular tri-chomes of several hyperaccumulating plants, includingArabidopsis halleri (Kupper et al., 2000; Zhao et al., 2000;Sarret et al., 2002) and Alyssum sp. (Broadhurst et al.,2004), and nonhyperaccumulating plants, includingBrassica juncea (Salt et al., 1995), Arabidopsis thaliana(Ager et al., 2003; Domınguez-Solıs et al., 2004), pump-kin (Cucurbita moschata; Iwasaki and Matsumura, 1999),and waterlily (Nymphaea sp.; Lavid et al., 2001), havebeen shown to sequester metals without apparent ex-cretion. Trichomes of calcicolous plants also store largeamounts of Ca. DeSilva et al. (1996) suggested that thiscould be a strategy to avoid the interference withstomatal functioning.

The mechanism of metal accumulation and excre-tion by trichomes and the binding environment ofmetals in these structures and their exudates arelargely unknown. The aim of this work is to clarifyseveral open questions that arose from our previousstudies on tobacco and to extend it to zinc (Zn). Thecation Cd21 is frequently associated with Ca21 in theenvironment because of their similar ionic radii. Doesthe production of Ca/metal grains occur for a cationicmetal of different size from Cd21, such as Zn21? If so,what is the influence of Ca21 supply on the metal

uptake, translocation, and formation of the grains?Then, what is the mineralogical nature of the excretedZn-containing grains, and what is the binding envi-ronment of Zn in the grains? This information isimportant because the chemical form of metals (inneror outer sphere complexes, coprecipitates with othermetals, etc.) determines their mobility and their pos-sible release in the environment. The most commonbiomineral formed by plants is CaC2O4 mono- anddihydrate. However, various forms of CaCO3, includ-ing amorphous CaCO3, calcite, aragonite, and vaterite,are also synthesized by plants (Arnott and Pautard,1970; Setoguchi et al., 1989). Another unknown is themechanism of formation of the grains, which aregenerally larger than the trichome cells themselves.And, finally, what is the relative importance of metalexcretion and accumulation in the leaf tissues?

To address these questions, tobacco plants weregrown in hydroponics and in vitro in the presence ofvarious concentrations of Ca and Zn. The effect of Caon the short-term (24 h) and long-term (2 and 5 weeks)accumulation of Zn and on the production of thegrains was studied. The endogenous accumulation ofZn in the top cells of trichomes was visualized underconfocal laser scanning microscopy (CLSM) after la-beling with Zinquin. The density of the trichomeswas estimated by examining fresh leaves by variablepressure scanning electron microscopy (VP-SEM), andisolated grains were examined by variable pressureand standard scanning electron microscopy coupledwith microanalysis (VP-SEM-EDX and SEM-EDX) andby a combination of three synchrotron-based tech-niques with a lateral resolution of a few micrometers.Specifically, the distribution of Ca and Zn in the grainswas imaged by micro x-ray fluorescence (mXRF), crys-talline phases were identified by micro x-ray dif-fraction (mXRD), and the binding environment of Znwas determined by Zn K-edge micro-extended x-rayabsorption fine structure (mEXAFS) spectroscopy.

RESULTS

Growth Retardation and Zn Accumulationin Tobacco Plants Treated with a Toxic Level

of Zn in Hydroponic Culture

Seed-derived tobacco plants were grown on hydro-ponic culture containing a high level of Zn (0.25 mM)and with and without supplement of 3 mM of Ca(Table I; Fig. 1). The Zn toxicity was monitored by thegrowth of shoots and roots and by chlorophyll con-tents (Table II). After 2 and 5 weeks of toxic Zn treat-ment, both shoot and root growth were severely affected,and the chlorophyll content was markedly reduced.The growth inhibition was more severe for roots thanfor shoots, especially after 5 weeks of exposure. Thesupplementation with 3 mM Ca suppressed the growthinhibition effect but did not restore chlorophyll level.These results suggest that Ca alleviates some (but notall) of the toxic effects of Zn.

Sarret et al.

1022 Plant Physiol. Vol. 141, 2006

The analysis of total Zn content showed that theCa supplement reduced the Zn accumulation inshoots after 24 h of Zn treatment (Table II). At theopposite, Zn accumulation was increased after 2 to 5weeks of the same Zn 1 Ca treatment. Consequently,the protective effect of Ca cannot be explained by acompetition between Ca and Zn resulting in a reducedZn uptake.

Growth and Trichome Density and Grain Productionin Tobacco Plants Cultured in Vitro

Growth retardation of tobacco plants culturedin vitro was similar to ex vitro culture in hydroponicmedium. However, Zn accumulation of in vitroplants was lower (Tables II and III), although thein vitro growing medium contained higher Znconcentration than the hydroponic medium.

Table I. Culture conditions and investigations

Culture

Conditions

Name of the

Treatment

Total Ca

Concentration in

the Substrate

Total Zn

Concentration in

the Substrate

Duration of

ExposureInvestigations Grain Name

mM mM

Hydroponic Control 0.28 0.08 3 1023 24 h, 2 and5 weeks

Zn contents in shoots; root and shootgrowth and chlorophyll content(2 and 5 weeks only); CLSM(2 weeks only)

Hydroponic Zn 0.28 0.25 24 h, 2 and5 weeks

Zn contents in shoots; root and shootgrowth and chlorophyll content(2 and 5 weeks only); CLSM(2 weeks only); SEM-EDX,mXRD, mXRF, and Zn mEXAFSon isolated grains (5 weeks only)

Zn1, Zn2

Hydroponic Zn 1 Ca 3.28 0.25 24 h, 2 and5 weeks

Zn contents in shoots; root and shootgrowth and chlorophyll content(2 and 5 weeks only); SEM-EDX,mXRD, mXRF, and Zn mEXAFSon isolated grains (5 weeks only)

ZnCa1, ZnCa2,ZnCa3, ZnCa4

Hydroponic Ca 3.28 0.08 3 1023 5 weeks SEM-EDX, mXRD, and mXRF onisolated grains

Ca1, Ca2

In vitro In vitro-control 3 0.03 3 weeks Shoot growth; size and numberof grains; SEM-EDX on isolatedgrains; trichome density

In vitro In vitro-Zn 3 1 3 weeks Shoot growth; size and number ofgrains; SEM-EDX on isolated grains;trichome density

In vitro In vitro-Zn 1 Ca 30 1 3 weeks Shoot growth; size and number ofgrains; SEM-EDX on isolated grains;trichome density

Figure 1. Tobacco plants grown for 2 weeks (A–C)and 5 weeks (D–F) in hydroponic medium containing0.08 mM Zn and 0.28 mM Ca (control), 0.25 mM Znand 0.28 mM Ca (Zn), and 0.25 mM Zn and 3.28 mM

Ca (Zn 1 Ca). Bars: A to C, 3.5 cm; D and E, 2.8 cm.

Excretion of Zinc by Tobacco Trichomes

Plant Physiol. Vol. 141, 2006 1023

This difference may result from a lower Zn bio-availability, respiration, and transpiration rates invitro.

In vitro plants were used for counting the number oftrichomes and isolating the grains because leaves oftobacco plants cultured ex vitro were more easily sub-jected to bacterial attack and contamination from aerosolparticles. Leaves of the plants treated with or without Znfor 3 weeks were observed under VP-SEM (Table III; Fig.2). There was no difference in trichome density for themature leaves. At the opposite, the young leaves ofplants exposed to Zn and Zn 1 Ca showed a more than2-fold increase of the density of long and short trichomerelative to the control (Fig. 2, A–C). No difference of leafsize was observed between the various treatments, sothis increase was real. This suggests a possible role of Znin the trichome development. Figure 2B showed that thelength and the volume of the trichomes were increasedupon Zn treatment, although epidermal cellsand stomatal opening looked smaller that those ofcontrol. A number of grains were observed on the topof both short and long trichomes, as seen in Figure 2C(arrows). For each treatment, the grains were collectedand observed by VP-SEM (Table III; Fig. 2, D–F). Theirnumber and their mean size increased in the followingorder: in vitro-Zn-Ca treatment . in vitro-Zn . in vitro-control (Table III; Fig. 2, D–F). We showed previouslythat exposure to 30 mM Ca without Zn (Ca treatment)did not enhance the production of the grains but stim-ulated the formation of intracellular Ca-containing crys-tals in leaves (Choi and Harada, 2005).

The same general trends in trichome density andgrain production were observed on plants grownhydroponically. The amount of Zn excreted per plantwas estimated based on the number and size of grainsper shoot and their Zn content calculated using theZAF (atomic number, absorption, fluorescence correc-tion) method (Goldstein et al., 1981). We found 4.1 and11.0 mg of Zn per plant under the in vitro-Zn andin vitro-Zn 1 Ca treatment, respectively, which corre-sponds to 9.2% and 16.9% of total Zn in the leaves,respectively. Note that these values reflect the grainspresent on the leaves after 3 weeks of exposure, not thetotal amount of Zn excreted during the experiment.

Confocal Imaging of Zn Accumulation in Trichome Cells

after Zinquin Labeling

Tobacco plants were grown hydroponically with0.25 mM Zn 1 0.28 mM Ca (Zn treatment) and 0.08 mM

Zn 1 0.28 mM Ca (control) for 2 weeks. Zn distributionon the leaf surfaces was investigated by CLSM aftertreatment with Zinquin (Fig. 3). Zinquin ethyl ester is alipo-soluble fluorescence dye that has been used todetect intracellular Zn ions in animal cells (Coyleet al., 1994). This study shows that it can be used onplant cells too. For control plants, a faint fluorescencewas detected in the trichomes (Fig. 3, A and C). ForZn-treated plants, Zn was preferentially accumulatedin head cells of both short and long trichomes (Fig. 3,B and D). Extracellular crystals were not present on thetop of the trichomes observed by CLSM because they

Table II. Plant growth, leaf chlorophyll contents, and Zn accumulation in shoots of tobacco grown under three conditions (culture conditionsdetailed in Table I)

Each value is the mean of three measurements on three different plants plus or minus the SD.

Treatment Height of Shoot Root Length

Chlorophyll Contents

Zn Accumulation in Shoot2 Weeks 5 Weeks

2 Weeks 5 Weeks 2 Weeks 5 Weeks Chlorophyll a Chlorophyll b Chlorophyll a Chlorophyll b 24 h 2 Weeks 5 Weeks

cm cm mg g21 FW mg g21 DW

Control 7.48 6 0.46 11.3 6 1.82 9.6 6 1.6 20.4 6 0.9 1.18 6 0.211 0.34 6 0.060 0.94 6 0.049 0.29 6 0.038 21.5 6 3.5 36.2 6 6.4 50.8 6 9.3

Zn 2.46 6 0.23 4.31 6 0.37 6.2 6 1.6 6.4 6 1.0 0.67 6 0.055 0.22 6 0.031 0.26 6 0.028 0.08 6 0.008 326.4 6 33.5 1,511.7 6 31.6 2,309.0 6 526.5

Zn 1 Ca 4.26 6 0.37 6.37 6 0.67 8.9 6 1.0 10.0 6 0.7 0.66 6 0.103 0.21 6 0.040 0.33 6 0.063 0.10 6 0.022 175.7 6 28.2 2,783.9 6 107.6 2,398.4 6 182.4

Table III. Shoot growth parameters and Zn content, number of trichomes, and grain production by the leaves of tobacco plants cultured in vitrofor 3 weeks under three conditions (culture conditions given in Table I)

Each value is the mean of three individual measurements plus or minus SD. The number of trichomes and grain production were determined byVP-SEM observations.

TreatmentHeight of

Shoot

Shoot Dry

Weight

Zn Accumulation

in Shoot

No. of Trichomes

per cm2 of Leaf

Grain Production and Zn Excretion

per Plant

Short

Trichome

Long

Trichome

No. of

Grains

Size of

Grains

Excreted

ZnaExcreted

Znb

cm mg mg g21 DW mm mg %

In vitro-control 8.7 6 0.56 171 6 12.3 91.7 6 12.2 21 6 3.7 14 6 2.3 127 6 34 26 6 11.2 – –In vitro-Zn 4.4 6 0.33 80 6 6.5 564.0 6 36.2 56 6 7.3 38 6 4.2 784 6 216 57 6 12.8 4.1 9.2In vitro-Zn 1 Ca 6.4 6 0.45 97 6 9.3 675.0 6 33.5 43 6 5.7 25 6 6.3 1,223 6 245 86 6 13.2 11.0 16.9

aExcreted Zn was calculated based on the number and size of excreted grains (Zn content was 2% in Zn grain, 1% in Zn 1 Ca grain, as estimatedby ZAF method). bPercentage of excreted Zn was calculated based on Zn amount in shoot.

Sarret et al.


fell off during the Zinquin treatment. Fluorescencewas also detected in the epidermal surfaces of theleaves but was always less dense than in the trichomecells (data not shown).

Zn Distribution Analysis by mXRF Spectrum

The distribution of Ca and Zn in the leaves oftobacco exposed to 0.25 mM Zn 1 3.28 mM Ca (Zn 1Ca treatment) was imaged by synchrotron-basedmXRF (Fig. 4). Zn was systematically more concen-trated in veins and trichomes, with the short trichomesbeing about 5 times richer than the long trichomes.Short trichomes were also rich in Ca but in the sameproportion as Zn (i.e. the Ca Ka:Zn Ka ratio equaledabout 1 in the two types of trichomes). No grains wereobserved at the top of the trichomes because they felloff during the sample preparation, such as freeze-drying and mounting and its rastering under the x-raybeam, but some were still detected as Zn hot-spots onthe leaf surfaces.

Morphology, Chemical Composition, and Solubilityof the Grains

Some grains present on the surface of a fresh leaffrom a Zn-treated plant were studied by VP-SEM-EDX(Fig. 5A), and other grains produced under various

conditions (in vitro and hydroponic culture with var-ious Ca and Zn concentrations; Table I) were collectedand studied by VP-SEM-EDX and SEM-EDX (Fig. 5,B–H). The size of the grains varied from 10 mm to150 mm in diameter for all treatments. A variety ofglobular to crystal-like shapes were observed. In mostcases, grains consisted in aggregates of small (,1 mm)particles, as previously observed for Cd21-treatedplants (Choi et al., 2001, 2004). The globular grainsgenerally were made of concentric layers (e.g. Fig. 5E).No particular relationship could be established be-tween the grain morphology and the type of treatment.

Ca was always the major element, even for plantsgrown on 0.28 mM Ca. In contrast, Zn was not detectedin the grains when its concentration in the nutrientsolution was low (Ca treatment, 0.08 mM Zn 1 3.28 mM

Ca). In grains from the Zn 1Ca-treated plants (0.25 mM

Zn 1 3.28 mM Ca), the Zn Ka fluorescence peak wassmall (Zn concentration ranged from 1–5 weight [wt]%, as estimated by the ZAF method), and it wasoccasionally more intense (Zn concentration rangedfrom 2–40 wt %) in grains from the Zn-treated plants(0.28 mM Ca 1 0.25 mM Zn). Minor elements includedoxygen (O), Mg, silicon (Si), P, S, chlorine (Cl), potassium(K), and Mn (carbon [C] could not be measured becausethe grains were coated with C). No particular relation-ship could be established between the concentration ofthe minor elements and the type of treatment, exceptfor Mn that was not detected in grains from the Zn1Ca

Figure 2. VP-SEM observation of the third leaf from the top (A–C) andof isolated grains (D–F) of tobacco grown in vitro for 3 weeks in amedium containing 0.03 mM Zn and 3 mM Ca (control), 1 mM Zn and3 mM Ca (Zn), and 1 mM Zn and 30 mM Ca (Zn 1 Ca). Bars: A to C,150 mm; D and E, 200 mm.

Figure 3. Distribution of Zn in long (A and B) and short (C and D)trichomes of tobacco obtained by confocal microscopy using Zinquinflorescent dye. Insets in A and B show optical microscope images.Plants were grown in hydropony in control (0.08 mM Zn and 0.28 mM

Ca) and Zn treatment (0.25 mM Zn and 0.28 mM Ca) for 2 weeks. The Znsignal is faint and almost evenly distributed in the trichomes of controlplants, whereas the trichomes of Zn-treated plants (particularly the longones) show a high concentration of Zn in the head cells. Bars: A and B,70 mm; C and D, 86 mm.



treatment. There was no morphological or composi-tional difference between grains observed directly onthe leaves using VP-SEM and grains isolated by waterextraction or using toothpicks. The solubility of thegrains in water was tested in a wide range of pH. Theywere insoluble between pH 4 and 12.5 and soluble inacidic conditions; they were dissolved within 10 min atpH 3 and within 2 min at pH 2. Therefore, the structureand the composition of the grains are supposed to bepreserved during the water extraction.

mXRD Analysis of the Grains

The grains produced by tobacco plants grown inhydroponics under the three treatments (Ca, Zn, andZn 1 Ca) were also analyzed by mXRD (Fig. 6). Five to10 grains per type of treatment were examined. Foreach grain, mXRD patterns from a 16 3 7 mm2 spot ofthe Ca-richest region (Ca treatment) or the Zn-richestregion (Zn and Zn 1 Ca treatment) and of the wholegrain were recorded. The point and average patternswere the same, except that the Debye rings for thesecond were less intense and longer (i.e. more powderlike). Therefore, either the Ca- and Zn-richest regionsare representative of the structural composition of theentire grains, or the other regions contain amorphousmaterial. Figure 6, A and B, shows the two-dimensionalmXRD patterns for two grains produced under the Catreatment (grains Ca1 and Ca2). The diameter of thesegrains is about 50 mm. Bragg reflections for grain Ca1consist of portions of Debye rings, indicating thatthe grain is composed of submicrometric crystals(Manceau et al., 2002). The intensities along each ring(constant Bragg angle) were integrated to obtain theone-dimensional XRD pattern presented in Figure 6C.The XRD peaks are slightly shifted to higher angles(i.e. smaller d values) relative to those of calcite(CaCO3), indicating smaller unit cell parameters. Therefined parameters were a5 4.929 A and c5 16.754 A,compared to a 5 4.9896 and c 5 17.0610 A for purecalcite (Supplemental Table I). The contraction of theunit cell is likely due to the substitution of smallercations for Ca. Based on mXRF analyses, Mn is apossible substituent for Ca. This particular grain wasnot analyzed by SEM-EDX, so the presence of lightelements (Z , 20) could not be tested. However, Mg

was frequently detected by SEM-EDX in other grainsfrom the same treatment (Fig. 5F; data not shown).As divalent Mn (0.67 A) and Mg (0.72 A) have asmaller ionic radius than Ca21 (1.00 A; Shannon, 1976),these two impurities are good candidate species forsubstitution at the Ca site. Assuming the presence ofonly one substitutional atom, the stoichiometry of theCa1 grain was calculated from the experimental a andc unit cell parameters using the Vegard law (West,1984). Briefly, this approach allows the determinationof the composition of a binary solid solution from thelinear regression between the composition and theunit cell parameters of the two pure end-members.Two independent calculations were performed witheither a or c. The difference in the stoichiometrycoefficients obtained by the two calculations was be-low 0.02 atom for all grains. For the Mg substitution,the end-members were calcite and magnesite(MgCO3), and the calculated formula for grain Ca1was Ca0.84Mg0.16CO3. For the Mn substitution, the end-members were calcite and rhodocrosite (MnCO3), andthe calculated formula was Ca0.75Mn0.25CO3. As thetwo impurities may be present in the grain, the pro-posed formula is Ca(0.84 2 0.09X)Mg[0.16(1 2 X)]Mn(0.25X)CO3,with X varying from 0 to 1. Whewellite (CaC2O4, H2O)was also detected in grain Ca1. This mineral hasseveral XRD peaks at the same position as thosefrom the substituted calcite crystallites, but the peakat Q 5 1.058 A21 (d 5 5.94 A) is characteristic of thisspecies, thereby allowing its unambiguous identifica-tion (Fig. 6C). This peak appears as a spotty feature onthe two-dimensional pattern (labeled W in Fig. 6A),which indicates that whewellite is coarsely crystallineat the scale of the x-ray probe (16 3 7 mm2). One XRDreflection was attributed to vaterite. This latter mineralwas more definitely identified in another experiment,in which tobacco was exposed to Cd21 (data notshown).

The mXRD pattern for grain Ca2 is spotty (Fig. 6B),which is indicative of coarse crystals. Most of x-rayreflections correspond to substituted calcite and have anintermediate position between those of pure calcite andgrain Ca1 (inset in Fig. 6C for the Bragg reflection 113),which suggests a lower degree of substitution relative tograin Ca1. The average structural formula of this grainwas calculated following the same procedure as forgrain Ca1, but, because of the smaller number of dif-fraction peaks, only the a parameter was refined (Sup-plemental Table I). Calculation yielded Ca(0.91 2

0.05X)Mg[0.09(1 2 X)]Mn(0.14X)CO3, with X varying from 0to 1. Fewer peaks, attributed to whewellite, weddellite(CaC2O4, 2 H2O), and vaterite (CaCO3), also weredetected (Fig. 6C).

Figure 7 shows mXRD data for two grains producedunder the Zn 1 Ca treatment (0.25 mM Zn 1 3.28 mM

Ca). These grains were rich in Ca and Zn, with the twoelements being heterogeneously distributed within thegrains (average Zn:Ca Ka ratio 5 7.5 and 1 for grainsZnCa1 and ZnCa2, respectively. The mXRD pattern forgrain ZnCa1 exhibits discontinuous rings as well as

Figure 4. Distribution of Ca and Zn determined by mXRF in a freeze-dried leaf of tobacco exposed to 0.25 mM ZnSO4 1 3.28 mM CaCl2 for5 weeks (Zn 1 Ca treatment). The long trichomes are rich in Ca,whereas the short trichomes are rich in Ca and Zn. The veins are richerin Zn than the leaf tissues. Bar 5 300 mm.

Sarret et al.


isolated spots (Fig. 7C). As for grains Ca1 and Ca2, theincomplete rings correspond to substituted calcite. Znis a likely substitute atom but not Mn because it wasnot detected in this grain (Fig. 7B). The presence of Mgwas not tested by SEM-EDX but is possible. Thechemical formula of this grain calculated with smith-sonite (ZnCO3) as Zn end-member and magnesite asMg end-member is Ca0.84(Mg,Zn)0.16CO3 (Supple-mental Table I). Mg21 and Zn21 have similar ionic

radii (0.72 and 0.75 A; Shannon, 1976) and, conse-quently, undergo similar reduction of the crystal latticeparameters. The isolated spots were attributed towhewellite. The mXRD pattern for grain ZnCa2 ex-hibited also incomplete Debye rings (data not shown).All peaks were attributed to Ca0.92(Mg,Zn)0.08CO3 crys-tals. The lower degree of substitution of calcite in grainZnCa2 compared to grain ZnCa1 is reflected in themXRD pattern by a smaller shift of its peaks relative to

Figure 5. A, Representative VP-SEM image and EDX spectrum of a grain present at the top of a trichome of tobacco plant grownin hydroponic culture under Zn treatment. B to H, VP-SEM (B–E) and SEM (F–H) images and EDX spectra of grains produced bytobacco plants grown in vitro (B–E) and hydroponically (F–H) under the various conditions listed in Table I. B and C, In vitro-control; D, in vitro-Zn; E, in vitro-Zn 1 Ca; F, Ca; G, Zn 1 Ca; H, Zn. EDX spectra were recorded at 20 kV.



calcite (inset in Fig. 7D for the Bragg reflections 110and 113).

Another form of CaCO3, aragonite, was identified ingrain ZnCa3 from the Zn 1 Ca treatment (Fig. 8).

This grain also contained Zn-substituted calcite(Ca0.93Zn0.07CO3). The continuous Debye rings ob-served in Figure 8A correspond to aragonite, andthe incomplete rings and speckles correspond to

Figure 6. A and B, mXRD patterns and mXRF spectra for grains Ca1 (A) and Ca2 (B) produced by the trichomes of a tobacco plantexposed to 3.28 mM Ca for 5 weeks (Ca treatment). The picture in A shows the grain mounted at the tip of a glass capillary. C,One-dimensional XRD patterns of the two grains. Grain Ca1 contains polycrystalline substituted calcite particles (SC) ofestimated composition Ca(0.84 2 0.09X)Mg[0.16(1 2 X)]Mn(0.25X)CO3, with X varying from 0 to 1, and coarse whewellite crystals (W,CaC2O4, H2O). Grain Ca2 contains polycrystalline calcite particles (SC) of estimated composition Ca(0.91 2 0.05X)Mg[0.09(1 2

X)]Mn(0.14X)CO3, with X varying from 0 to 1, and whewellite (W, CaC2O4, H2O), weddellite (Wd, CaC2O4, 2 H2O), and vaterite (V,CaCO3) coarse crystals. The shift to lower d values (higher Q values) of the diffraction peaks for grain Ca1 relative to grain Ca2indicates that the former contains more impurities (i.e. higher substitution degree). Inset, Enlargement of the 113 reflections of thetwo grains. The peak position of pure calcite is indicated by a vertical bar.

Figure 7. A, Ca and Zn distributions for grain ZnCa1 produced by tobacco exposed to 0.25 mM Zn 1 3.28 mM Ca for 5 weeks.B, mXRF spectrum of the same grain recorded at an incident energy of 17 keV on the Zn-richest spot. C, mXRD pattern of thewhole grain. D, One-dimensional XRD patterns for grains ZnCa1 and ZnCa2 produced in the same conditions. Grain ZnCa1contains substituted calcite [unlabeled peaks, Ca0.84(Mg,Zn)0.16CO3] and whewellite (W, CaC2O4, H2O), and ZnCa2 containssubstituted calcite [all peaks, Ca0.92(Mg,Zn)0.08CO3] only. Similar to grains Ca1 and Ca2, the higher the degree of substitution, thelarger is the shift of the XRD peaks toward the low d values (or high Q values) relative to pure calcite (inset).

Sarret et al.


substituted calcite. Thus, aragonite occurs as nano-particles, whereas substituted calcite occurs as submi-crometric crystals. Another grain (ZnCa4) containedCa0.87Zn0.13CO3 (Supplemental Table I; SupplementalFig. 1). For ZnCa3 and ZnCa4, Zn was the onlysubstitute atom because neither Mg nor Mn wasdetected by mXRF and SEM-EDX (Figs. 8B and 5Gfor the EDX spectra; mXRF spectra not shown).

Several grains produced under the Zn treatmentcontained (Mg,Zn)-substituted calcite (e.g. grain Zn1;Supplemental Table I; Supplemental Fig. 2), and othergrains (e.g. grain Zn2; Fig. 5H) did not produce hklreflections, although they also contained Zn and Ca.The amorphous nature of these grains was confirmedby Zn-EXAFS (see below) and by Ca-x-ray absorptionnear-edge structure spectroscopy (data not shown).

In conclusion, submicrometer-sized calcite crystalssubstituted by Zn, Mg, and Mn were the most frequentmineral species. Other minerals identified include, bydecreasing order of abundance, whewellite (alwayscoarsely crystalline), weddellite, aragonite, and vater-ite. ZnC2O4 was never detected. Zn-containing grainswere also analyzed by Zn K-edge mEXAFS spec-troscopy to confirm the presence of Zn-substitutedcalcite, to gain more insight on the nature of the amor-phous or poorly crystallized Zn species, and to deter-mine the proportions of all Zn phases in case of mixture.

Zn K-Edge mEXAFS Analysis of the Grains

Figure 9 shows the Zn mEXAFS spectra of the Zn-richest regions for the grains. Spectra with severalfrequencies (e.g. ZnCa1 and ZnCa2) are indicative of along-range ordered Zn-binding environment, such as

that in a mineral structure, whereas spectra dominatedby a single frequency (e.g. Zn2) are indicative of ashort-range ordered Zn environment. Spectra ZnCa3,ZnCa4, and Zn1 are somehow intermediate betweenthe two previous sets of spectra, suggesting a mixtureof Zn species. Data analysis was done by linear com-bination fits using an extended database of Zn refer-ence compounds containing Zn-bearing and Zn-sorbedminerals, Zn precipitates, and Zn complexed to simpleorganic acids and complex biopolymers (Sarret et al.,2004; Guine et al., 2006). One- to three-componentsimulations were tested. All spectra were reproducedcorrectly with two components (Fig. 9; Table IV).Adding a third one did not improve the fit qualitysignificantly, as indicated by the normalized sum-squares residual parameter (NSS) because its valuedecreased by less than 10% upon adding a thirdcomponent.

Spectra ZnCa1 and ZnCa2 show a marked similarityto that of Zn-substituted calcite, the major differencebeing a smaller amplitude of the unknowns. Accord-ingly, the best one-component fits for these two sam-ples were obtained with Zn-substituted calcite (TableIV; dashed lines for ZnCa1 in Fig. 9). The wavefrequencies and overall shapes of the unknownswere satisfactorily reproduced by this model com-pound but not the amplitude at low k. Adding asecond component greatly improved the simulations.Fits of equivalent quality were obtained with eitherZn-humic acid complex (Zn-organic acids in Table IV)or Zn-sorbed hydroxylapatite (Zn-sorbed phosphate)as second component. Zn complexes with simpleorganic acids (oxalate, citrate, malate, etc.) weretested also but did not provide satisfactory fits. The

Figure 8. A, Two-dimensional XRD pattern for grain ZnCa3 produced by tobacco exposed to 0.25 mM Zn 1 3.28 mM Ca for5 weeks. B, EDX spectrum recorded at 20 kV. C, One-dimensional XRD pattern. Arrowed peaks correspond to substituted calcite[Ca0.93(Mg,Zn)0.07CO3], peaks noted by a dashed line correspond to substituted calcite and aragonite, and all other peakscorrespond to aragonite. Inset, SEM image of the grain.



Zn-humic acid complex reference was used as a proxyfor Zn bonded to multiple functional groups, includ-ing carboxyl, hydroxyl, and phenol groups. This com-ponent species may correspond to organic compoundsexcreted by the trichomes. Crystalline Zn phosphatecompounds, including Zn phosphate tetrahydrate(hopeite and parahopeite) and Zn phosphate dihy-drate, did not provide good fits to the data. Fortechnical reasons, grains ZnCa1 and ZnCa2 were not

analyzed by SEM-EDX; consequently, the presence ofP could not be verified by chemical analysis. However,this element was frequently detected in grains fromthe three treatments, as shown for the Ca treatment inFigure 5F. Therefore, Zn-organics and Zn phosphateforms are equally probable species in grains ZnCa1and ZnCa2. The presence of Zn in calcite is consistentwith mXRD results for the two grains. Note that calcitegrains may also contain Mg since this element wasoften detected by SEM-EDX.

Spectra ZnCa3, ZnCa4, and Zn1 were simulatedcorrectly by a mixture of Zn-substituted calcite andZn-organic acids (NSS 5 7.2, 7.9, and 4.8, respectively;Table IV). The fractional amount of Zn-substitutedcalcite (15%–23%) is lower than that in grains ZnCa1and ZnCa2 (40%–53%), in agreement with the spectraldifferences of the two sets of grains (see above).Simulations with Zn-sorbed phosphate instead ofZn-organic acids were much weaker (NSS 5 0.136,0.120, and 0.098, respectively). This result is consistentwith the absence of P in ZnCa4 and Zn1 (Fig. 5G;Supplemental Fig. 2B), and its trace amounts in ZnCa3(Fig. 8B). Again, the occurrence of Zn-substitutedcalcite in grains ZnCa3, ZnCa4, and Zn1 is consistentwith mXRD results.

The incorporation of Zn in the aragonite crystallitesfrom grain ZnCa3 was not tested because of the lack ofZn-substituted aragonite reference. However, this spe-cies is unlikely, or at most minor, for two reasons. First,mXRD data showed that this carbonate species is notsubstituted, in contrast to calcite. Second, the EXAFSspectrum of Zn-substituted aragonite would be differ-ent from that of Zn-substituted calcite because thesetwo carbonate polymorphs do not have the samestructure. The fact that the sum of the contributionsfrom individual reference spectra to all sample spectra(except ZnCa4) is 100% 6 10% adds further credibilityto the determined speciation. The lower sum forZnCa4 (i.e. 80%) probably results from an overabsorp-tion effect (Manceau et al., 2002). This effect decreasesthe amplitude of the measured signal without modi-fying the relative contributions of the componentspectra.

Spectrum Zn2 was reproduced best with a combi-nation of Zn-sorbed silica and Zn-organic acids. Thepresence of Si was confirmed by SEM-EDX (Fig. 5H).Zn-sorbed phosphate also provided a good fit to thedata (NSS 5 2.9), but this model was rejected becausethe grain did not contain P. The absence of Zn-substituted calcite in this grain is consistent with thelack of diffraction peaks on the mXRD pattern.

In conclusion, the EXAFS analysis engenders confi-dence in the presence of Zn-substituted calcite thanksto the unique spectral signature of this species and tothe consistency in the interpretations of the spectro-scopic and diffraction data. In addition, this species islogically more abundant in the Zn 1 Ca treatment thanin the Zn treatment. EXAFS spectroscopy is less sen-sitive to Zn-sorbed silica, Zn-sorbed phosphate, andZn-organic acids as a result of the short-range order of

Figure 9. Zn bulk EXAFS spectra for selected Zn reference compounds,and Zn mEXAFS spectra for grains produced by tobacco exposed for5 weeks to 0.25 mM Zn 1 3.28 mM Ca (ZnCa1, ZnCa2, ZnCa3, andZnCa4) and to 0.25 mM Zn (Zn1 and Zn2). Solid line, Experimentaldata; dashed line, one-component fit (for ZnCa1 only); dotted line, two-component fit.

Sarret et al.


Zn, multiplicity of Zn-binding environments, andpresence of light elements (C, Si, and P) in the secondcoordination sphere of Zn in these species. Despitethese difficulties, their occurrence suggested by EX-AFS data is supported by the detection of Si and P bySEM-EDX and by the fact that tobacco trichomesexcrete organic compounds.

DISCUSSION

The positive effect of Ca on metal tolerance shownhere has been observed in other plant species (Chenet al., 2000; Samecka-Cymerman and Kempers, 2001).Generally, this effect is attributed to a modification ofthe speciation of the metal in the nutrient solutionand its competition with Ca for the plant uptake. Inthis study, increasing the Ca supply inhibited the Znuptake after 24 h but not after 2 and 5 weeks ofexposure. Consequently, the positive effect of Ca onZn tolerance results from a cooperative and not aninhibition mechanism between the two elements. Ca

and Zn supplements increased the density of thetrichomes and led to the production of Ca/Zn-containing grains (Table III; Fig. 2, C and F), suggest-ing that this excretion is a way to eliminate the excessof Zn from the plant tissues.

The mineralogical analysis of the grains showed thatthey contained calcium carbonates (primarily Zn-substituted calcite and, secondarily, vaterite and ara-gonite) and CaC2O4 mono- and dihydrate. CaC2O4 is arather common mineral in higher plants. In tobacco, ithas been found in the vacuoles of specialized leaf cells,called idioblasts (Bouropoulos et al., 2001), and its pres-ence in the trichome cells has been suggested (Wagneret al., 2004). This mineral has been shown to sequesterheavy metals in some higher plants (Mazen, 2004, andrefs. therein) and lichens (Sarret et al., 1998, and refs.therein). This study shows that this is not the case inthe grains excreted by tobacco trichomes. CaCO3 is lessusual in plants. Cystoliths, which are calcified bodiesformed in specialized cells called lithocysts, have beendescribed in some plants (Setoguchi et al., 1989). Thesestructuresconsistpredominantly of amorphous CaCO3,

Table IV. mEXAFS and mXRD results

Grain

Proportion of Zn

Species (%) Determined

by Linear Combination Fitting

the mEXAFS Spectra Sum NSSbElements Detected by

SEM-EDX and/or

mXRFa

Minerals Identified

by mXRD

Zn-Substituted

Calcite

Zn-Organic

Acids

Zn-Sorbed

Silica

Zn-Sorbed

Phosphate

%

Grains from the Ca treatmentc

Ca1 Ca, Mn (elements lighterthan Ca not analyzed)

Subst. calcite(Ca(0.84 2 0.09X)Mg[0.16(1 2 X)]Mn

(0.25X)CO3), whewellite(CaC2O4, H2O)

Ca2 Ca, Mn (elements lighterthan Ca not analyzed)

Subst. calcite(Ca(0.91 2 0.05X)Mg[0.09(1 2 X)]

Mn(0.14X)CO3), whewellite(CaC2O4, H2O), weddellite(CaC2O4, 2 H2O), vaterite (CaCO3)

Grains from the Ca 1 Zn treatmentc

ZnCa1 53 – – 53 23.6d Zn, Ca (elements lighterthan Ca not analyzed)

Subst. calcite(Ca0.84(Mg,Zn)0.16CO3),whewellite (CaC2O4, H2O)

40 50 – – 90 8.9d

44 – – 49 93 9.5ZnCa2 59 – – – 59 18.9 Zn, Ca (elements lighter

than Ca not analyzed)Subst. calcite

(Ca0.92(Mg,Zn)0.08CO3)50 39 – – 89 11.3d

53 – – 38 91 11.7ZnCa3 22 78 – – 100 7.2d Zn, Ca, Si, O Subst. calcite

(Ca0.93Zn0.07CO3),aragonite (CaCO3)

ZnCa4 23 57 – – 80 7.9d Zn, Ca, Si, O Subst. calcite(Ca0.87Zn0.13CO3)

Grains from the Zn treatmentc

Zn1 15 73 – – 88 4.8d Zn, Ca, K, Cl, Si, Mg, O Subst. calcite (Ca0.92(Mg,Zn)0.08CO3)Zn2 – 37 79 – 115 2.7d Zn, Ca, K, Cl, Si, Mg, O

aCarbon could not be analyzed because the grains were coated with this element. bResidual between fit and experimental data: NSS5 S [k3 x(k) 2k3 x(k)fit]

2/S [k3 x(k)]2 3 100. cCa and Zn concentrations given in Table I. dSimulations shown in Figure 9.



along with subordinate amounts of calcite and vaterite,and their function is unclear. This study reports theformation of several types of CaCO3 by tobacco andthe sequestration of Zn specifically in calcite. Otherchemical forms of Zn found in the grains were Zn-organic acids, Zn-sorbed silica, and Zn-sorbed phos-phate. Si is not a component of the nutrient solution.This element might come from glass vessels used toprepare the solutions. The proportion of Zn-substituted calcite relative to the other forms increasedwith Ca concentration in the nutrient medium.

Although the mechanism of formation of the grainsremains unclear, some speculations can be offered.Considering the diameter of the grains (10–150 mm)and the diameter of the trichome plasmodesmata oftobacco (Waigmann et al., 1997), they are obviouslyformed externally. However, Zn and Ca are bioaccu-mulated within the trichomes before their exudationas shown by mXRF, and more precisely in the top cellsfor Zn, as shown by CLSM (Ca not studied by CLSM).The fact that each grain is heterogeneous in chemicalcomposition and consists in an aggregate of submi-crometer-sized particles might be due to an excretionof Ca, Zn, and the other elements in liquid form, fol-lowed by precipitation in contact with air. The pre-dominance of calcite relative to the other CaCO3polymorphs further supports this hypothesis. Indeed,in a supersaturated solution of Ca21 and CO3

22, amor-phous CaCO3 precipitates first, then transforms tovaterite, and finally to calcite, which is the mostthermodynamically stable CaCO3 polymorph (Oginoet al., 1990). In the presence of Mg21, the formation ofaragonite is favored at the expense of calcite. There-fore, following the definition of Lowenstam and Wei-ner (1989), the crystallization process seems to bebiologically induced rather than biologically con-trolled.

In the perspective of metal exposure through smok-ing, an enhanced excretion of metal-containing grainsduring the plant growth and the removal of the grainsduring the cigarette manufacturing process might helpto decrease the Zn and Cd content in the tobacco prod-ucts. Studies on the genetics of trichome differentia-tion and excretion (Hallahan et al., 2000; Wagner et al.,2004) should contribute to better understand, and pos-sibly enhance, metal excretion through the trichomes.In contrast, in the perspective of phytoextraction, theproduction and the release of metal-containing grainsto the soil should be avoided to optimize the metalcontent in the harvested shoots. In this study, Zn se-questered amount in the grains of in vitro plantsrepresented 9.2% and 16.9% in Zn- and Zn 1 Ca-treatedplants, respectively, as estimated from the number andchemical composition of the grains. To estimate morerealistically the proportion of excreted Zn, one shouldmonitor the production and composition of the grainsduring the whole growth period and consider the totalcontent of Zn in leaf at the time of the harvest.

Finally, this study illustrates the potential of micro-focused x-ray techniques in the fields of biominerali-

zation and bioremediation. When combined withmolecular biology and biochemical tools, these tech-niques should provide key information on the strate-gies developed by plants to cope with metal toxicityand maintain metals homeostasis.

MATERIALS AND METHODS

Plant Cultures

The culture conditions and the experimental techniques are listed in Table

I. For the hydroponic cultures, seeds were germinated on solid medium-filled

PCR tubes and transferred after 3 weeks to 1.5-L pots (three plants per pot)

filled with one-tenth-strength Hoagland medium. Fe was supplied as N,N-di-

(2-hydroxybenzoyl)-ethylendiamine N,N#-di-acetic acid (5 mM; Weber et al.,

2004). To avoid insect attack and dust contamination, plants were grown at

22�C in a closed culture box in a growth chamber with 16-h-light/8-h-dark

cycle. After 3 weeks in the Hoagland medium, plants were maintained in this

medium containing 0.08 mM ZnSO4 and 0.28 mM CaCl2 (control medium) or

transferred to a medium containing 0.25 mM ZnSO4 and 0.28 mM CaCl2 (Zn

treatment) or 0.25 mM ZnSO4 and 3.28 mM CaCl2 (Zn 1 Ca treatment; Table I).

After 2 or 5 weeks, shoots and roots were harvested separately. The height of

the shoots and the root length were measured. Aliquots of shoots were freeze-

dried, weighted, and digested in 10 mL of 60% nitric acid by a microwave

system (mPREP-A; MLS GmbH) applying the following gradient program: 2.5

min, 700 W, 75�C, 15 bar; 8 min, 500 W, 130�C, 25 bar; 12 min, 1,000 W, 200�C,

45 bar; and 24 min, 1,000 W, 200�C, 45 bar. Zn concentrations in the digests

were measured by inductively coupled plasma-atomic emission spectrometry

with a Perkin-Elmer OPTIMA 300DV. Chlorophyll was extracted with 80%

acetone and measured spectrophotometrically as described by Delfine et al.

(1999). Each analysis was done on three replicates.

For the short-term (24-h) exposure, plants were precultured in the Hoagland

medium for 5 weeks and transferred to the control, Zn, and Zn 1 Ca media

(Table I). After 24 h, plants were harvested and analyzed as describe above.

For in vitro culture, seed-derived plants at about 5-cm height were trans-

ferred in 300-mL glass culture bottles containing one-third-strength Mura-

shige and Skoog medium solidified with 0.7% agar and 1% Suc and exposed to

0.03 mM ZnSO4 and 3 mM CaCl2 (in vitro-control), 1 mM ZnSO4 and 3 mM CaCl2

(in vitro-Zn treatment), and 1 mM ZnSO4 and 30 mM Ca (in vitro-Zn 1 Ca

treatment; Table I). Plants were cultivated in a growth room at 25�C under a

16-h photoperiod (35 mmol m22 s21 white fluorescent tubes). Trichome den-

sities were determined under VP-SEM (S-3500N; Hitachi) by counting the

short and long trichomes on the third leaf from the top and dividing these

numbers by the leaf area. To collect the grains, plants were plunged in pure

water in 50-mL tubes and vortexed for a few seconds. The supernatant was

carefully removed, and the grains were collected at the bottom with a pipette.

Another isolation procedure was tested: grains were carefully collected using

toothpicks under a stereomicroscope (Olympus SZX11-ST). Thereafter, grains

were dried in vacuo (Speed Vac SC100; Savant Instruments), counted under

VP-SEM (S-3500N; Hitachi), and analyzed chemically by EDX. To test the

solubility of isolated grains, solutions with wide range of pH (2.0, 3.0, 4.0, 5.0,

7.0, 9.0, and 12.5) were prepared by adjusting the pH of ultrapure water with

HCl or NaOH. Isolated grains were put in water drops on the slide glass and

then observed the time-lapse solubility under light microscope.

Also, ex vitro hydroponic culture of tobacco plants was undertaken to check

that the production of Ca/Zn-containing grains was not an artifact of in vitro

conditions and to obtain grains for morphological, mineralogical, and spectro-

scopic characterization. After 5 weeks of hydroponic culture in the control, Zn,

Zn 1 Ca, and Ca media (Table I), grains were collected using the water

extraction procedure and kept under dry atmosphere prior to analyses. Leaf

segments were also harvested and directly observed under VP-SEM-EDX or

plunged in liquid nitrogen and freeze-dried for elemental mapping by mXRF.

VP-SEM-EDX and SEM-EDX

Hydrated leaves and isolated grains from the in vitro and hydroponic

cultures were glued on an aluminum stub, then mounted in a chamber stage

after cooling to 220�C and observed by VP-SEM-EDX using a Hitachi S-3500N

fitted with a Horiba EMAX-7000 x-ray detector (Kuboki and Wada, 1995). The

chamber pressure was 30 Pa and the accelerating voltage 15 kV. Isolated grains

Sarret et al.


from the hydroponic cultures were also analyzed by SEM-EDX using a JEOL

JSM 840A microscope fitted with a Kevex Si (Li) x-ray detector. The grains

were attached on a C stub with C tape and coated with C. The analyses were

performed under vacuum at an accelerating voltage of 15 kV.

CLSM

For visualization of intracellular Zn, leaf segments of plants grown in

hydropony in 0.25 mM Zn 1 0.28 mM Ca control (Zn treatment) and 0.08 mM

Zn 1 0.28 mM Ca (control) for 2 weeks were immersed in 13 PBS buffer

(137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4)

containing 25 mM Zinquin ethyl ester ([ethyl (2-methyl-8-p-toluenesulphona-

mido-6-quinolyloxy) acetate]; Biotium) for 1 h at 25�C. Leaf segments were

washed once with PBS buffer and mounted on glass slides and examined

with a CLSM system (LSM510 META NLO; Carl Zeiss) with Plan-Neofluar

103 (0.3 NA) or 203 (0.5 NA) water-immersion lens. Zinquin florescence

was determined at an excitation wavelength of 368 nm with an UV Ar laser

(80 mW) at an emission wavelength of 490 nm. Confocal fluorescence and

concurrent differential interference contrast images were taken, and digital

images were processed using AxioCam HR with LSM 5 images software.

mXRF, mXRD, and mEXAFS

The experiments were performed on beamline 10.3.2 of the Advanced Light

Source, Berkeley, CA (Marcus et al., 2004). For the mXRF and mEXAFS mea-

surements, the beam was focused down to 5 3 5mm and the x-ray fluorescence

was measured with a seven-element Ge detector. For themXRD measurements,

the diffraction patterns were recorded with a 1,024 3 1,024 pixels CCD camera

at 17 keV incident x-ray energy with a 16 3 7 mm2 beam size. Isolated tobacco

grains were mounted at the top of a glass capillary (0.2 mm in diameter), and the

capillary fixed on a motorized x-y-z stage. The grains were cooled to 150 K with

a Cryostream cooler (Oxford 611) during measurements to minimize any

potential beam damage. First, a mXRD pattern of the whole grain was recorded

by scanning it under the beam while acquiring the data. Then, a point mXRD

pattern, a mXRF spectrum, and a Zn K-edge EXAFS spectrum (if Zn present)

were recorded successively on the Ca- and Zn-richest spots. mXRF elemental

maps from the freeze-dried leaf segments were recorded at room temperature

in mounting the sample directly on the x-y-z stage.

XRD and EXAFS Data Treatment

The two-dimensional XRD patterns were calibrated using an alumina

standard and integrated to one-dimensional patterns for peak assignment

with the JCPDS database (http://www.icdd.com). The stoichiometry of the

Mg-, Mn-, and Zn-substituted crystals was estimated by refining the unit cell

parameters a and c over the [10–33] � 2u angular range ([1.3–4.0] A interval)

using the Ufit software (M. Evain, 1992), and then by applying the Vegard law

(West, 1984) using calcite and magnesite (MgCO3) as end-members for the

Ca-Mg solid solution; calcite and rhodocrosite (MnCO3) as end-members for

the Ca-Mn solid solution; and calcite and smithsonite (ZnCO3) as end-

members for the Ca-Zn solid solution.

EXAFS spectra were extracted using the standard procedure and then

simulated by linear combinations of reference spectra from an extended

spectral library (Sarret et al., 2004; Guine et al., 2006). The spectrum for Zn-

substituted calcite was provided by R.J. Reeder (Reeder et al., 1999; Elzinga

and Reeder, 2002). Zn-sorbed silica was prepared by adding 40 mg of

Zn(NO3)2 4 H2O (2.55 mM Zn) to a suspension containing 0.5 g of silica

(aerosil 200; Degussa) pre-equilibrated at pH 6.0, stirring the suspension for 2 h

at pH 6.0, centrifugating it, and freeze-drying the solid phase. The final Zn

content was 2 wt %. The Zn-humic acid and Zn-reacted hydroxylapatite

references were described previously (Sarret et al., 1997; Panfili et al., 2005).

ACKNOWLEDGMENTS

We thank Delphine Tisserand and Martine Lanson (Laboratoire de Geo-

physique Interne et Tectonophysique, France), Sebastien Pairis (Laboratoire

de Cristallographie, France), Sylvia Kruger (Leibniz-Institut fur Pflanzenbio-

chemie, Germany), and Jeong-Yeon Han (Kangwon National University,

Korea) for their technical assistance. VP-SEM-EDX and CLSM analyses were

performed at Korea Basic Science Institute, Chunchon, South Korea. We

acknowledge R.J. Reeder for sharing the Zn-calcite EXAFS spectrum, and

Prof. Dierk Scheel and Dr. Dieter Neumann (Leibniz-Institut fur Pflanzen-

biochemie) for fruitful discussions.

Received April 28, 2006; revised April 28, 2006; accepted May 22, 2006;

published May 26, 2006.

LITERATURE CITED

Ager FJ, Ynsa MD, Domınguez-Solıs JR, Lopez-Martın MC, Gotor C,

Romero LC (2003) Nuclear micro-probe analysis of Arabidopsis thaliana

leaves. Nucl Inst Meth Phys Res B 210: 401–406

Arnott HJ, Pautard FGE (1970) Calcification in plants. In H Schraer, ed,

Biological Calcification: Cellular and Molecular Aspects. Appleton-

Century-Crofts, New York, pp 375–446

Bouropoulos N, Weiner S, Addadi L (2001) Calcium oxalate crystals in

tomato and tobacco plants: morphology and in vitro interactions of

crystal-associated macromolecules. Chem Eur J 7: 1881–1888

Bringezu K, Lichtenberger O, Leopold I, Neumann D (1999) Heavy metal

tolerance of Silene vulgaris. J Plant Physiol 154: 536–546

Broadhurst CL, Chaney RL, Angle JS, Maugel TK, Erbe EF, Murphy CA

(2004) Simultaneous hyperaccumulation of nickel, manganese, and

calcium in Alyssum leaf trichomes. Environ Sci Technol 38: 5797–5802

Chen ZS, Lee GJ, Liu JC (2000) The effects of chemical remediation

treatments on the extractability and speciation of cadmium and lead in

contamination soils. Chemosphere 41: 235–242

Choi YE, Harada E (2005) Roles of calcium and cadmium on Cd-containing

intra- and extracellular formation of Ca crystals in tobacco. J Plant Biol

48: 113–119

Choi YE, Harada E, Kim GH, Yoon ES, Sano H (2004) Distribution of

elements on tobacco trichomes and leaves under cadmium and sodium

stresses. J Plant Biol 47: 75–82

Choi YE, Harada E, Wada M, Tsuboi H, Morita Y, Kusano T, Sano H (2001)

Detoxification of cadmium in tobacco plants: formation and active

excretion of crystals containing cadmium and calcium through tri-

chomes. Planta 213: 45–50

Coyle P, Zalewski PD, Philcox JC, Forbes IJ, Ward AD, Lincoln SF,

Mahadevan I, Rofe AM (1994) Measurement of zine in hepatocytes by

using a fluorescent probe, Zinquin: relationship to metallothionein and

intracellular zine. Biochem J 303: 781–786

Delfine S, Alvino A, Villiani MC, Loreto F (1999) Restrictions to carbon

dioxide conductance and photosynthesis in spinach leaves recovering

from salt stress. Plant Physiol 119: 1101–1106

deSilva DLR, Hetherington AM, Mansfield TA (1996) Where does all the

calcium go? Evidence of an important regulatory role for trichomes in

two calcicoles. Plant Cell Environ 19: 880–886

Domınguez-Solıs JR, Lopez-Martın MC, Ager FJ, Ynsa MD, Romero LC,

Gotor C (2004) Increased cysteine availability is essential for cadmium

tolerance and accumulation in Arabidopsis thaliana. Plant Biotechnol J 2:

469–476

Elzinga EJ, Reeder RJ (2002) X-ray absorption spectroscopy study of Cu21

and Zn21 adsorption complexes at the calcite surface: implications for

site-specific metal incorporation preferences during calcite crystal

growth. Geochim Cosmochim Acta 66: 3943–3954

Fleisher RL, Parungo FP (1974) Aerosol particles on tobacco trichomes.

Nature 250: 158–159

Goldstein JI, Newbury DE, Echlin P, Joy DC, Fiori C, Lifshin E (1981)

Scanning Electron Microscopy and X-Ray Microanalysis. Plenum Press,

New York

Guine V, Spadini L, Sarret G, Muris M, Delolme C, Gaudet J, Martins J

(2006) Zinc sorption to three gram-negative bacteria: combined titration,

modeling and EXAFS study. Environ Sci Technol 40: 1806–1813

Hallahan D, Gray J, Callow J, editors (2000) Advances in Botanical

Research: Plant Trichomes. Academic Press, London

Iwasaki K, Matsumura A (1999) Effect of silicon on alleviation of manga-

nese toxicity in pumpkin (Cucurbita moschata Duch cv. Shintosa). Soil Sci

Plant Nutr 45: 909–920

Kuboki K, Wada M (1995) The variable pressure SEM with the cooling

stage. Hitachi Instrum News 27: 20–25

Kupper H, Kroneck PMH (2005) Heavy metal uptake by plants and

cyanobacteria. In A Sigel, H Sigel, R Sigel, eds, Metal Ions In Biological

Systems, Vol 44. Marcel Dekker, New York, pp 92–142



Kupper H, Lombi E, Zhao FJ, McGrath SP (2000) Cellular compartmen-

tation of cadmium and zinc in relation to other elements in the hyper-

accumulator Arabidopsis halleri. Planta 212: 75–84

Lavid N, Barkay Z, Tel-Or E (2001) Accumulation of heavy metals in

epidermal glands of the waterlily (Nymphaeaceae). Planta 212:

313–322

Lowenstam H, Weiner S (1989) On Biomineralization. Oxford University

Press, New York, Oxford

Lugon-Moulin N, Zhang M, Gadani F, Rossi L, Koller D, Krauss M,

Wagner GJ (2004) Critical review of the science and options for reducing

cadmium in tobacco (Nicotiana tabacum L.) and other plants. Adv Agron

83: 111–180

MacFarlane GR, Burchett M (1999) Zinc distribution and excretion in the

leaves of the grey mangrove, Avicennia marina (Forsk.) Vierh. Environ

Exp Bot 41: 167–175

Manceau A, Marcus MA, Tamura N (2002) Quantitative speciation of heavy

metals in soils and sediments by synchrotron X-ray techniques. In P

Fenter, M Rivers, N Sturchio, S Sutton, eds, Applications of Synchrotron

Radiation in Low-Temperature Geochemistry and Environmental Sci-

ence, Vol 49. Reviews in Mineralogy and Geochemistry. Mineralogical

Society of America, Washington, DC, pp 341–428

Marcus MA, MacDowell AA, Celestre R, Manceau A, Miller T, Padmore

HA, Sublett RE (2004) Beamline 10.3.2 at ALS: a hard X-ray microprobe

for environmental and materials sciences. J Synchrotron Radiat 11: 239–247

Mazen AMA (2004) Calcium oxalate deposits in leaves of Corchorus

olitotius as related to accumulation of toxic metals. Russ J Plant Physiol

51: 281–285

Neumann D, zur Nieden U, Lichtenberger O, Leopold I (1995) How does

Armeria maritima tolerate high heavy metal concentrations? J Plant

Physiol 146: 704–717

Ogino T, Suzuki T, Sawada K (1990) The rate and mechanism of poly-

morphic transformation of calcium carbonate in water. J Cryst Growth

100: 159–167

Panfili F, Manceau A, Sarret G, Spadini L, Kirpichtchikova T, Bert V,

Laboudigue A, Marcus MA, Ahamdach N, Libert MF (2005) The effect

of phytostabilization on Zn speciation in a dredged contaminated

sediment using scanning electron microscopy, X-ray fluorescence,

EXAFS spectroscopy and principal components analysis. Geochim

Cosmochim Acta 69: 2265–2284

Reeder RJ, Lamble GM, Northrup PA (1999) XAFS study of the coordina-

tion and local relaxation around Co21, Zn21, Pb21 and Ba21 trace

elements in calcite. Am Mineral 84: 1049–1060

Rodriguez E, Healey PL, Mehta I, editors (1983) Biology and Chemistry of

Plant Trichomes. Plenum Press, New York

Salt DE, Prince RC, Pickering IJ, Raskin I (1995) Mechanisms of cadmium

mobility and accumulation in Indian mustard. Plant Physiol 109:

1427–1433

Samecka-Cymerman A, Kempers AJ (2001) Concentration of heavy metals

and plant nutrients in water, sediments and aquatic macrophytes of

anthropogetic lakes (former open cut brown coal mines) differing in

stage of acidification. Sci Total Environ 281: 87–98

Sarret G, Balesdent J, Bouziri L, Garnier JM, Marcus MA, Geoffroy N,

Panfili F, Manceau A (2004) Zn speciation in the organic horizon of a

contaminated soil by micro X-ray fluorescence, micro and powder

EXAFS spectroscopy and isotopic dilution. Environ Sci Technol 38:

2792–2801

Sarret G, Manceau A, Cuny D, Van Haluwyn C, Deruelle S, Hazemann JL,

Soldo Y, Eybert-Berard L, Menthonnex JJ (1998) Mechanism of

lichen resistance to metallic pollution. Environ Sci Technol 32:

3325–3330

Sarret G, Manceau A, Hazemann JL, Gomez A, Mench M (1997) EXAFS

study of the nature of zinc complexation sites in humic substances as a

function of Zn concentration. J Phys IV 7: 799–802

Sarret G, Saumitou-Laprade P, Bert V, Proux O, Hazemann JL, Traverse A,

Marcus MA, Manceau A (2002) Forms of zinc accumulated in the

hyperaccumulator Arabidopsis halleri. Plant Physiol 130: 1815–1826

Setoguchi H, Okazaki M, Suga S (1989) Calcification in higher plants with

special reference to cystoliths. In R Crick, ed, Origin, Evolution, and

Modern Aspects of Biomineralization in Plants and Animals. Plenum

Press, New York, pp 409–418

Shannon RD (1976) Revised effective ionic radius and systematic studies of

interatomic distances in halides and chalcogenides. Acta Crystallogr

A 32: 751–767

Shepherd RW, Bass WT, Houtz RL, Wagner GJ (2005) Phylloplanins of

tobacco are defensive proteins deployed on aerial surface by short

glandular trichomes. Plant Cell 17: 1851–1861

Song W-Y, Sohn EJ, Martinoia E, Lee YJ, Yang Y-Y, Jasinski M, Forestier C,

Hwang I, Lee Y (2003) Engineering tolerance and accumulation of lead

and cadmium in transgenic plants. Nat Biotechnol 21: 914–919

Stephens WE, Calter A, Newton J (2005) Source and health implications of

high toxic metal concentrations in illicit tobacco products. Environ Sci

Technol 39: 479–488

Wagner GJ, Wang E, Shepherd RW (2004) New approaches for studying

and exploiting an old protuberance, the plant trichome. Ann Bot (Lond)

93: 3–11

Waigmann E, Turner A, Peart J, Roberts K, Zambryski P (1997) Ultra-

structural analysis of leaf trichome plasmodesmata reveals major dif-

ferences from mesophyll plasmodesmata. Planta 203: 75–84

Weber M, Harada E, Vess C, von Roepenack-Lahaye E, Clemens S (2004)

Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis

halleri roots identifies nicotianamine synthase, a ZIP transporter and

other genes as metal hyperaccumulation factors. Plant J 37: 269–281

West A (1984) Solid State Chemistry and Its Applications. Wiley, New York

Zhao F, Lombi E, Breedon T, McGrath SP (2000) Zinc hyperaccumulation

and cellular distribution in Arabidopsis halleri. Plant Cell Environ 23:

507–514

Sarret et al.


trichomes of tobacco excrete zinc as zinc-substituted...

Documents